Nonlinear mixed-modelling discriminates the effect of chemicals and their mixtures on zebrafish behavior


Zebrafish (Danio rerio) early-life stage behavior has the potential for high-throughput screening of neurotoxic environmental contaminants. However, zebrafish embryo and larval behavioral assessments typically utilize linear analyses of mean activity that may not capture the complexity of the behavioral response. Here we tested the hypothesis that nonlinear mixed-modelling of zebrafish embryo and larval behavior provides a better assessment of the impact of chemicals and their mixtures. We demonstrate that zebrafish embryo photomotor responses (PMRs) and larval light/dark locomotor activities can be fit by asymmetric Lorentzian and Ricker-beta functions, respectively, which estimate the magnitude of activity (e.g., maximum and total activities) and temporal aspects (e.g., duration of the responses and its excitatory periods) characterizing early life-stage zebrafish behavior. We exposed zebrafish embryos and larvae to neuroactive chemicals, including isoproterenol, serotonin, and ethanol, as well as their mixtures, to assess the feasibility of using the nonlinear mixed-modelling to assess behavioral modulation. Exposure to chemicals led to distinct effects on specific behavioral characteristics, and interactive effects on temporal characteristics of the behavioral response that were overlooked by the linear analyses of mean activity. Overall, nonlinear mixed-modelling is a more comprehensive approach for screening the impact of chemicals and chemical mixtures on zebrafish behavior.


Zebrafish (Danio rerio) have proven to be an excellent model for ecotoxicological applications1, in part due to their tractability in laboratory settings, high fecundity, and well-understood and precisely-timed ontogeny2. The zebrafish is also an excellent model to study chemical effects on developmental programming, as well as early life-stage behavior3,4,5. For instance, as early as 30 hours post fertilization (hpf), zebrafish embryos exhibit a short photomotor response (PMR) involving a spike in movement over a 10 s period4. The PMR has high plasticity to drug exposure and has been used for high-throughput screening of neuroactive chemicals6,7. Also, after hatch (>72 hpf) the larvae exhibit distinct locomotor activity to alternating dark and light photoperiods8. Locomotor activity is supressed when larvae are held in an illuminated environment, while a switch to complete darkness evokes a spike in locomotor activity lasting around 15 min5,8. Recent studies suggest that this larval behavior may be disrupted by environmental contaminants5,8,9.

Although animal behavioral endpoints hold promise in ecotoxicology for risk assessments, they are not widely used by regulatory agencies. A major challenge in using animal behavior for risk assessment involves the wide-ranging responses that require careful assessment and selection of the most appropriate statistical analyses for biological/ecological relevance10. Animal activity is often measured by averaging the values temporally and/or spatially to make them amenable for linear analyses11,12. However, that greatly simplifies behavioral analyses because activity changes are seldom linear. This is exemplified by the nonlinear embryo PMR and larval locomotor activity profiles (i.e., asymmetric peak- and hump-shaped respectively6,8). These activity profiles reveal behavioral characteristics, including quickness, duration and maximum intensity, which are analogous to common parameters of nonlinear functions13.

Nonlinear mixed-modelling includes both fixed-effects (i.e., model parameters – phenotypical characteristics) and random-effects (i.e., within-subject effects), and allows for phenotype comparisons, while controlling for repeated measurements of activity during the behavioral trials14,15. Also, the inclusion of an interaction term in the fixed-effects component of the model allows for testing interactive effects16. Against this backdrop, our objective was to test the feasibility of using nonlinear mixed-modelling to assess the effects of chemicals either alone or as mixtures on zebrafish embryo and larval activity. Specifically, we tested the hypothesis that nonlinear mixed-modelling is a more sensitive representation of chemical effects and their interactions on early life-stage zebrafish behavior compared to linear modelling of mean activity. As a proof of concept, we carried out zebrafish embryo PMR and larval locomotor activity trials with fish exposed to model chemicals that either stimulate or suppress embryo and larvae activity. We also co-exposed embryos and larvae to a mixture of the stimulant and suppressor to demonstrate the capacity of nonlinear mixed-modelling in testing and describing mixture toxicity.


Zebrafish maintenance and embryo collection

Adult zebrafish (Tupfel long fin strain) were cultured in 10 L polypropylene tanks at 28.5 °C, pH 7.6, and 740 µS conductivity on recirculating systems (Pentair Aquatic Habitats, Apopka, Florida). The recirculating systems were housed in an animal care facility at the University of Calgary with a 14 h:10 h light:dark daily light cycle. Animals were fed with ZieglerTM adult zebrafish diet (Pentair) and live Artemia (San Francisco Bay Brand, Inc, Newark, CA, USA) in the morning and evening respectively. Zebrafish were bred and the early life-stages maintained in E3 medium17 at 28.5 °C as described previously5. The animal maintenance and all experiments were approved by the animal care committee at the University of Calgary (AC17-0079) and were in accordance with the Canadian Council on Animal Care guidelines.

Zebrafish PMR

We followed the protocol outlined in Kokel et al.6 with slight modifications. We 3D-printed a custom light emitting diode (LED)-array for use with a ZebraBox behavioral acquisition system (Viewpoint Life Sciences, Montreal, QC, Canada) that allowed for multiple configurations of LEDs (Super Bright LEDs, St. Louis, Missouri, USA) that could be activated with a remote switch. PMR trials were 30 s in duration and were carried out in total darkness, with the exception of two 1 s light pulses at 10 and 20 s6, and were recorded at 30 frames per second. Embryos were dechorionated at 24 hpf in 1 g L−1 Pronase (Sigma) and transferred (6 embryos per well) to the center 48 wells of a 96-well plate (Greiner, Sigma) with 225 µL of embryo medium per well. Only the center 48 wells were used to maximize magnification and resolution of video acquisition for activity measurements. Preliminary work identified that exposure to the β-adrenergic receptor agonist, isoproterenol, and ethanol stimulated and supressed the PMR, respectively. The treatments included 100 µM isoproterenol, 2% ethanol or a combination of both and the exposures began 30 min before each PMR trial. Final well volume was 300 uL following additions of chemical stocks. Treatments were randomly assigned by plate column, and each plate contained 2 columns (i.e., 16 wells) of control, isoproterenol, ethanol, and isoproterenol-ethanol treatments. A total of 360 embryos per treatment were used for the PMR experiments. The PMR trials began at 32 hpf with 20 min dark acclimation periods between plates. Embryo activity was quantified as Δ pixel intensity from each frame.

Zebrafish larval locomotor activity

Larvae were transferred to each well of a 96-well plate at 80 hpf along with 225 µL of embryo medium and maintained overnight at 28.5 °C. Preliminary work identified that exposure to isoproterenol and serotonin suppressed and stimulated larval locomotor activity, respectively. The next day each well received either 20 µM isoproterenol, 100 µM serotonin or a combination of both and each plate contained 24 wells/larvae per treatment (a total of 3 plates). Final well volume was 300 uL following additions of chemical stocks. Immediately following the chemical exposure, plates were transferred to the Zebrabox and behavioral trials commenced as described previously18, following the lighting regime of Emran et al.8. The Zebrabox system includes backlighting in the visible spectrum from 0% to 100% intensity. Our lighting regime included 30 min at 0% intensity (i.e., dark adaptation), 30 min at 100% intensity, and a final 30 min at 0% intensity. Larval activity was calculated as total distance travelled every 30 s as described previously5,18.